An inherent challenge within cancer research is the cataloguing of fundamental information on what is generally fatal metastatic disease within vital organs. Treating metastatic progression remains a formidable task due to an inability to monitor specific differential molecular adaptations that allow for the cancer to survive and thrive within different tissue types. Hence, an important consideration is the divergence of the metastatic cancer cells growing in visceral organs from the primary tumor. To address this problem, we have established isogenic human metastatic breast cancer cell lines, which are representative of the common metastatic sites of lung, bone, liver, and brain observed in breast cancer patients. Our preliminary biological evaluations of these new metastatic cell lines strongly indicated that they exhibit site-to-site phenotypic distinctions that ultimately reflect genetic diversity as well as drug resistance differences. In addition, we have preliminary label-free Raman spectroscopy and metabolomic characterizations that provide objective evidence of the distinct biochemical differences that specifically identify each of the cell lines. Importantly, we have carried out label-free Raman spectroscopy on human breast cancer tissue microarray slides and obtained defined spectral biochemical signatures that differentiate normal from metastatic breast tissues. The importance of this finding is that we have now established objective molecular methods that will permit the identification of unique signatures of breast cancer metastatic lesions. The central hypothesis is that identification of molecular signatures that can be assigned to site-specific breast cancer metastatic lesions will facilitate optimal treatment strategies to increase overall survival. In this application, three specific Aims have been proposed to test the above hypothesis. In specific Aim 1, we will establish Raman spectral signatures of newly established isogenic organ-specific metastatic breast cancer cells in vitro. In specific Aim 2, we will validate Raman spectral signatures in preclinical models of breast cancer metastasis and in matched primary and metastatic human breast cancer samples. In specific Aim 3, we will determine chemotherapeutic efficacy of FDA approved oncological drugs in a site-specific manner based on Raman spectral signatures.